Tsvetan R. Bachvaroff

Broad Phylogenomic Sampling and the Sister Lineage of Land Plants

The tremendous diversity of land plants all descended from a single charophyte green alga that colonized the land somewhere between 430 and 470 million years ago. Six orders of charophyte green algae, in addition to embryophytes, comprise the Streptophyta s.l. Previous studies have focused on reconstructing the phylogeny of organisms tied to this key colonization event, but wildly conflicting results have sparked a contentious debate over which lineage gave rise to land plants. The dominant view has been that ‘stoneworts,’ or Charales, are the sister lineage, but an alternative hypothesis supports the Zygnematales (often referred to as “pond scum”) as the sister lineage. In this paper, we provide a well-supported, 160-nuclear-gene phylogenomic analysis supporting the Zygnematales as the closest living relative to land plants. Our study makes two key contributions to the field: 1) the use of an unbiased method to collect a large set of orthologs from deeply diverging species and 2) the use of these data in determining the sister lineage to land plants. We anticipate this updated phylogeny not only will hugely impact lesson plans in introductory biology courses, but also will provide a solid phylogenetic tree for future green-lineage research, whether it be related to plants or green algae.

Strain variation in Karlodinium veneficum (Dinophyceae): toxin profiles, pigments, and growth characteristics.

Karlodinium veneficum (D. Ballant.) J. Larsen strains, 16 from the U.S. Atlantic eastern seaboard and two from New Zealand (CAWD66 and CAWD83), were used to characterize toxin profiles during batch culture. All 18 strains were determined as the same species based on ITS sequence analyses, a positive signal in a chloroplast real‐time PCR assay and pigment composition. Five karlotoxin 1 (KmTx 1) containing strains were analyzed from the Chesapeake Bay, and 10 karlotoxin 2 (KmTx 2) strains were analyzed from Florida to North Carolina. One strain (MD5) from the Chesapeake Bay produced no detectable toxin. The two cultures from New Zealand contained both novel karlotoxins with lower masses and earlier elution times. Toxin type did not change during batch culture, although the KmTx phenotype did change in some strains under extensive (months) phototrophic growth in replete media. KmTx cell quota did not change during batch culture for most strains. The mass spectrum for every KmTx examined showed a pattern of multiple coeluting congeners within each HPLC peak, with masses typically differing by 16 amu. KmTx congeners tested showed nearly a 500‐fold range in specific hemolytic activity, with KmTx 1 (typically occurring at lower cellular levels) most hemolytic and CAWD66 toxin least hemolytic, while KmTx 2 and the CAWD83 toxin had similar intermediate specific activity. Despite morphological, genetic, and photopigment indicators consistent with species homogeneity among the 18 strains of K. veneficum, the high degree of toxin variability suggests different functional roles among strains that likely coexist in situ.

Molecular phylogeny of ocelloid-bearing dinoflagellates (Warnowiaceae) as inferred from SSU and LSU rDNA sequences

BackgroundDinoflagellates represent a major lineage of unicellular eukaryotes with unparalleled diversity and complexity in morphological features. The monophyly of dinoflagellates has been convincingly demonstrated, but the interrelationships among dinoflagellate lineages still remain largely unresolved. Warnowiid dinoflagellates are among the most remarkable eukaryotes known because of their possession of highly elaborate ultrastructural systems: pistons, nematocysts, and ocelloids. Complex organelles like these are evolutionary innovations found only in a few athecate dinoflagellates. Moreover, the taxonomy of warnowiids is extremely confusing and inferences about the evolutionary history of this lineage are mired by the absence of molecular phylogenetic data from any member of the group. In this study, we provide the first molecular phylogenetic data for warnowiids and couple them with a review of warnowiid morphological features in order to formulate a hypothetical framework for understanding character evolution within the group. These data also enabled us to evaluate the evolutionary relationship(s) between warnowiids and the other group of dinoflagellates with complex organelles: polykrikoids.ResultsMolecular phylogenetic analyses of SSU and LSU rDNA sequences demonstrated that warnowiids form a well-supported clade that falls within the more inclusive Gymnodinium sensu stricto clade. These data also confirmed that polykrikoids are members of the Gymnodinium sensu stricto clade as well; however, a specific sister relationship between the warnowiid clade and the polykrikoid clade was unresolved in all of our analyses. Nonetheless, the new DNA sequences from different isolates of warnowiids provided organismal anchors for several previously unidentified sequences derived from environmental DNA surveys of marine biodiversity.ConclusionComparative morphological data and molecular phylogenetic data demonstrate that the polykrikoid and the warnowiid clade are closely related to each other, but the precise branching order within the Gymnodinium sensu stricto clade remains unresolved. We regard the ocelloid as the best synapomorphy for warnowiids and infer that the most recent common ancestor of polykrikoids and warnowiids possessed both nematocysts and photosynthetic plastids that were subsequently lost during the early evolution of warnowiids. Our summary of species and genus concepts in warnowiids demonstrate that the systematics of this poorly understood group is highly problematic and a comprehensive revision is needed.

Alveolate phylogeny inferred using concatenated ribosomal proteins.

ABSTRACT. Dinoflagellates and apicomplexans are a strongly supported monophyletic group in rDNA phylogenies, although this phylogeny is not without controversy, particularly between the two groups. Here we use concatenated protein‐coding genes from expressed sequence tags or genomic data to construct phylogenies including “typical” dinophycean dinoflagellates, a parasitic syndinian dinoflagellate, Amoebophrya sp., and two related species, Oxyrrhis marina, and Perkinsus marinus. Seventeen genes encoding proteins associated with the ribosome were selected for phylogenetic analysis. The dataset was limited for the most part by data availability from the dinoflagellates. Forty‐five taxa from four major lineages were used: the heterokont outgroup, ciliates, dinoflagellates, and apicomplexans. Amoebophrya sp. was included in this phylogeny as a sole representative of the enigmatic marine alveolate or syndinian lineage. The atypical dinoflagellate O. marina, usually excluded from rDNA analyses due to long branches, was also included. The resulting phylogenies were well supported in concatenated analyses with only a few unstable or weakly supported branches; most features were consistent when different lineages were pruned from the tree or different genes were concatenated. The least stable branches involved the placement of Cryptosporidium spp. within the Apicomplexa and the relationships between P. marinus, Amoebophrya sp., and O. marina. Both bootstrap and approximately unbiased test results confirmed that P. marinus, Amoebophrya sp., O. marina, and the remaining dinoflagellates form a monophyletic lineage to the exclusion of Apicomplexa.

PHYLOGENY OF FOUR DINOPHYSIACEAN GENERA (DINOPHYCEAE, DINOPHYSIALES) BASED ON rDNA SEQUENCES FROM SINGLE CELLS AND ENVIRONMENTAL SAMPLES(1).

Dinoflagellates are a highly diverse and environmentally important group of protists with relatively poor resolution of phylogenetic relationships, particularly among heterotrophic species. We examined the phylogeny of several dinophysiacean dinoflagellates using samples collected from four Atlantic sites. As a rule, 3.5 kb of sequence including the nuclear ribosomal genes SSU, 5.8S, LSU, plus their internal transcribed spacer (ITS) 1 and 2 regions were determined for 26 individuals, including representatives of two genera for which molecular data were previously unavailable, Ornithocercus F. Stein and Histioneis F. Stein. In addition, a clone library targeting the dinophysiacean ITS2 and LSU sequences was constructed from bulk environmental DNA from three sites. Three phylogenetic trees were inferred from the data, one using data from this study for cells identified to genus or species (3.5 kb, 28 taxa); another containing dinoflagellate SSU submissions from GenBank and the 12 new dinophysiacean sequences (1.9 kb, 56 taxa) from this study; and the third tree combing data from identified taxa, dinophysiacean GenBank submissions, and the clone libraries from this study (2.1 kb, 136 taxa). All trees were congruent and indicated a distinct division between the genera Phalacroma F. Stein and Dinophysis Ehrenb. The cyanobionts containing genera Histioneis and Ornithocercus were also monophyletic. This was the largest molecular phylogeny of dinophysoid taxa performed to date and was consistent with the view that the genus Phalacroma may not be synonymous with Dinophysis.

Revision of the family Duboscquellidae with description of Euduboscquella crenulata n. gen., n. sp. (Dinoflagellata, Syndinea), an intracellular parasite of the ciliate Favella panamensis Kofoid & Campbell.

Recent recognition that tintinnids are infected by dinophycean as well as syndinean parasites prompts taxonomic revision of dinoflagellate species that parasitize these ciliates. Long overlooked features of the type species Duboscquella tintinnicola are used to emend the genus and family Duboscquellidae, resulting in both taxa being moved from the Syndinea to the Dinophyceae. Syndinean species previously classified as Duboscquella are relocated to Euduboscquella n. gen., with Euduboscquella crenulata n. sp. as the type. As an endoparasitic species, E. crenulata shares with its congeners processes associated with intracellular development and sporogenesis, but differs from closely related species in nuclear and cortical morphology of the trophont, including a distinctively grooved shield (= episome) that imparts a crenulated appearance in optical section. In addition, E. crenulata produces three morphologically distinct spore types, two of which undergo syngamy to form a uninucleate zygote. The zygote undergoes successive division to produce four daughter cells of unequal size, but that resemble the nonmating spore type.

Tintinnophagus acutus n. g., n. sp. (Phylum Dinoflagellata), an Ectoparasite of the Ciliate Tintinnopsis cylindrica Daday 1887, and Its Relationship to Duboscquodinium collini Grassé 1952

ABSTRACT. The dinoflagellate Tintinnophagus acutus n. g., n. sp., an ectoparasite of the ciliate Tintinnopsis cylindrica Daday, superficially resembles Duboscquodinium collini Grassé, a parasite of Eutintinnus fraknoii Daday. Dinospores of T. acutus are small transparent cells having a sharply pointed episome, conspicuous eyespot, posteriorly positioned nucleus with condensed chromosomes, and rigid form that may be supported by delicate thecal plates. Dinospores attach to the host via a feeding tube, losing their flagella, sulcus, and girdle to become spherical or ovoid cells. The trophont of T. acutus feeds on the host for several days, increasing dramatically in size before undergoing sporogenesis. Successive generations of daughter sporocytes are encompassed in an outer membrane or cyst wall, a feature not evident in trophonts. Tintinnophagus acutus differs from D. collini in host species, absence of a second membrane surrounding pre‐sporogenic stages, and failure to differentiate into a gonocyte and a trophocyte at the first sporogenic division. Phylogenetic analyses based on small subunit (SSU) ribosomal DNA (rDNA) sequences placed T. acutus and D. collini in the class Dinophyceae, with T. acutus aligned loosely with Pfiesteria piscicida and related species, including Amyloodinium ocellatum, a parasite of fish, and Paulsenella vonstoschii, a parasite of diatoms. Dubosquodinium collini nested in a clade composed of several Scrippsiella species and Peridinium polonicum. Tree construction using longer rDNA sequences (i.e. SSU through partial large subunit) strengthened the placement of T. acutus and D. collini within the Dinophyceae.

Expressed Sequence Tags from Amoebophrya sp. Infecting Karlodinium veneficum: Comparing Host and Parasite Sequences

ABSTRACT. Parasitic dinoflagellates of the genus Amoebophrya play important roles in the ecology of estuaries and open ocean environments. Little is known of the cell and molecular biology of Amoebophrya, but the genus is intermediate on phylogenetic trees between apicomplexans and typical dinophycean dinoflagellates. Here, we constructed four cDNA libraries, from different stages after infecting the host, Karlodinium veneficum, with Amoebophrya sp. These libraries were used to generate 898 expressed sequence tags (ESTs), with sequences attributed to either the host or parasite, based on AT bias, codon usage, and occurrence during infection. Overall, 209 sequences were attributable to the parasite and 685 to the host. The 50 putative parasite sequences with good protein matches in GenBank were used to find the same protein from host ESTs. For 26 genes, both host and parasite sequences were identified, of which 20 encoded ribosomal proteins. PCR for seven predicted parasite and two host genes were used to confirm attributions. The most common host and parasite ESTs were compared to see if multiple gene copies were present. The host plastocyanin gene had multiple sequence variants, but parasite rps27a contained only one polymorphism, likely due to an amplification error. Amplification, cloning, and sequencing of five parasite protein‐coding genes suggested that the parasite has a single sequence for each gene, but three host genes were found to have multiple variants. The genome of Amoebophrya sp. infecting K. veneficum appears to have an organization more similar to other eukaryotes than to the tandem gene arrangements found in dinoflagellates.

Structural, functional, and evolutionary aspects of galectins in aquatic mollusks: From a sweet tooth to the Trojan horse.

Galectins constitute a conserved and widely distributed lectin family characterized by their binding affinity for β-galactosides and a unique binding site sequence motif in the carbohydrate recognition domain (CRD). In spite of their structural conservation, galectins display a remarkable functional diversity, by participating in developmental processes, cell adhesion and motility, regulation of immune homeostasis, and recognition of glycans on the surface of viruses, bacteria and protozoan parasites. In contrast with mammals, and other vertebrate and invertebrate taxa, the identification and characterization of bona fide galectins in aquatic mollusks has been relatively recent. Most of the studies have focused on the identification and domain organization of galectin-like transcripts or proteins in diverse tissues and cell types, including hemocytes, and their expression upon environmental or infectious challenge. Lectins from the eastern oyster Crassostrea virginica, however, have been characterized in their molecular, structural and functional aspects and some notable features have become apparent in the galectin repertoire of aquatic mollusks. These including less diversified galectin repertoires and different domain organizations relative to those observed in vertebrates, carbohydrate specificity for blood group oligosaccharides, and up regulation of galectin expression by infectious challenge, a feature that supports their proposed role(s) in innate immune responses. Although galectins from some aquatic mollusks have been shown to recognize microbial pathogens and parasites and promote their phagocytosis, they can also selectively bind to phytoplankton components, suggesting that they also participate in uptake and intracellular digestion of microalgae. In addition, the experimental evidence suggests that the protozoan parasite Perkinsus marinus has co-evolved with the oyster host to be selectively recognized by the oyster hemocyte galectins over algal food or bacterial pathogens, thereby subverting the oysters innate immune/feeding recognition mechanisms to gain entry into the host cells.

Diversity of Eukaryotic Translational Initiation Factor eIF4E in Protists

The greatest diversity of eukaryotic species is within the microbial eukaryotes, the protists, with plants and fungi/metazoa representing just two of the estimated seventy five lineages of eukaryotes. Protists are a diverse group characterized by unusual genome features and a wide range of genome sizes from 8.2 Mb in the apicomplexan parasite Babesia bovis to 112,000-220,050 Mb in the dinoflagellate Prorocentrum micans. Protists possess numerous cellular, molecular and biochemical traits not observed in “text-book” model organisms. These features challenge some of the concepts and assumptions about the regulation of gene expression in eukaryotes. Like multicellular eukaryotes, many protists encode multiple eIF4Es, but few functional studies have been undertaken except in parasitic species. An earlier phylogenetic analysis of protist eIF4Es indicated that they cannot be grouped within the three classes that describe eIF4E family members from multicellular organisms. Many more protist sequences are now available from which three clades can be recognized that are distinct from the plant/fungi/metazoan classes. Understanding of the protist eIF4Es will be facilitated as more sequences become available particularly for the under-represented opisthokonts and amoebozoa. Similarly, a better understanding of eIF4Es within each clade will develop as more functional studies of protist eIF4Es are completed.